Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F2/00—Processes of polymerisation
  • C08F2/44—Polymerisation in the presence of compounding ingredients, e.g. plasticisers, dyestuffs, fillers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/829—Alcaligenes
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/848—Escherichia
  • Y10S435/849—Escherichia coli
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/859—Micrococcus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/874—Pseudomonas
  • Y10S435/875—Pseudomonas aeruginosa
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/886—Streptomyces
  • Y10S435/897—Streptomyces griseus

Definitions

• This invention relates to a process for preparing 6-aminopenicillanic acid. More particularly, it relates to the production of 6-aminopenicillanic acid (hereinafter referred to simply as "6-APA") by enzymatic reaction of an immobilized penicillin amidase-producing microorganism with penicillin.
• 6-APA 6-aminopenicillanic acid
• 6-APA is prepared by enzymatic deacylation of penicillin, i.e., by the reaction of penicillin with penicillin amidase or a penicillin amidase-producing microorganism.
• this method is disadvantageous for a large scale production.
• 6-APA produced according to this method is contaminated with the enzyme, microbial cells, nutrient sources and/or proteins. Accordingly, additional steps of removing the enzyme, microbial cells and/or other contaminants from the product are required to recover 6-APA in high purity.
• the reaction solution is boiled and/or acidified to destroy the enzyme or microorganism, and precipitates of the enzyme or microorganism are filtered off.
• 6-APA is prepared by chemical deacrylation of penicillin.
• penicillin is reacted with triphenylmethyl chloride to give triphenylmethyl ester of penicillin.
• Said ester is treated with phosphorus trichloride.
• the triphenylmethyl ester of N imino -chloro-penicillin thus obtained is reacted with methanol to give the corresponding N imino -methoxy-penicillin ester.
• 6-APA is obtained by hydrolyzing said N imino -methoxy-penicillin ester with aqueous ammonia.
• this chemical method is also disadvantageous because of lengthy procedures involved therein and/or low yield of 6-APA to be obtained.
• 6-APA can be prepared by the steps of polymerizing at least one acryloyl monomer in an aqueous suspension of a penicillin amidase-producing microorganism, and subjecting the resultant immobilized penicillin amidase-producing microorganism to enzymatic reaction with penicillin.
• Preferred examples of the penicillin amidase-producing microorganisms which are employed in the present invention include Escheichia coli ATCC 9637, Escherichia coli ATCC 11105, Streptomyces griseus IFO (Institute for Fermentation, Osaka, Japan) 3355, Nocardia gardneri ATCC 9604, Micrococcus roseus IFO 3764, Pseudomonas aeruginosa OUT (Faculty of Technology, Osaka University, Japan) 8252 and Alkaligenes faecalis OUT 8025. All of these microorganisms are publicly available from the above-mentioned depositories.
• the present invention is not limited to the use of these specific microorganisms, but includes within its scope the use of all of penicillin amidase-producing microorganisms such as those belonging to the genera of Escherichia, Streptomyces, Nocardia, Alkaligenes, Micrococcus and Pseudomonas.
• Suitable amount of the penicillin amidase-producing microorganism which is employed in the present invention is 0.32 to 3.2 g, especially 0.95 to 1.9 g, per g of the acryloyl monomer or monomers used.
• the polymerization reaction of the present invention serves to tightly entrap each of the microorganisms into the lattice of the polymer thereby affording high enzymatic activity for a long period of time.
• the polymerization reaction of the present invention can be carried out in the presence of a polymerizaton initiator and a polymerization accelerator.
• a polymerizaton initiator Potassium persulfate, ammonium persulfate vitamin B 2 and Methylene Blue are suitable as polymerization initiators.
• ⁇ -(dimethylamino)-propionitrile and N,N,N',N'-tetramethylethylenediamine are employed as polymerizaton accelerators.
• Suitable amount of polymerization initiator to be added to the aqueous suspension of the penicillin amidase-producing microorganism are 5 to 50 mg per g of the acryloyl monomer or monomers.
• Suitable amounts of polymerization accelerator to be added are 1.5 to 15 mg per g of the acryloyl monomer or monomers. It is preferred to carry out the reaction of 0° to 50°C, especially 20° to 40°C. The reaction may be completed within 10 to 60 minutes.
• the acryloyl monomers which are suitable for use in the present invention include acryloylamide, N,N'-lower alkylene-bis-acryloylamide, bis(acryloylamidomethyl)ether and N,N'-di-acryloyl-ethylene-urea(N,N'-acryloyl-imidazolidine-2-one).
• the penicillin amidase-producing microorganism with a polymer obtained from one or two monomers mentioned above, particularly with a copolymer of acryloylamide and an acryloyl monomer selected from the group consisting of N,N'-lower alkylene-bis-acryloylamide, bis(acryloylamidomethyl)ether and N,N'-di-acryloyl-ethyleneurea.
• N,N'-methylene-bis-acryloylamide and N,N'-propylene-bis-acryloylamide are preferably employed as the N,N'-lower alkylene-bis-acryloylamide.
• a suitable amount of N,N'-lower alkylene-bis-acryloylamide, bis(acryloylamidemethyl)ether or N,N'-acryloyl-ethylene-urea which is used to copolymerize with acryloylamide is 5 to 160 mg, especially 15 to 130 mg, more specifically 25 to 80 mg, per g of acryloylamide.
• the resultant immobilized penicillin amidase-producing microorganism is granulated by passing it through a sieve to form particles of 2.5 to 5.5 mm, especially 3 to 4 mm in diameter.
• 6-APA can be prepared by enzymatic reaction of the immobilized penicillin amidase-producing microorganism with penicillin.
• the enzymatic reaction is carried out at 25° to 50°C, especially at 30° to 45°C. It is preferred to carry out reaction at a pH of 7 to 9.5, especially at pH 8 to 9.
• Any known penicillins can be used as a substrate of the present invention. Representative examples of said penicillins are shown by the following formula: ##EQU1## wherein R is an alkyl, alkenyl or aryl-alkyl group, or a group of the formula: R 1 --X--CH 2 --, R 1 is alkyl, alkenyl or aryl, and X is oxygen or sulfur.
• the known penicillins which are employed in the present invention include Penicillin G(benzylpenicillin), Penicillin K(n-heptylpenicillin), Penicillin X(p-hydroxybenzylpenicillin), Penicillin F(2-pentenylpenicillin), Dihydropenicillin F(n-amylpenicillin), Penicillin AT(allylmercaptomethylpenicillin), Penicillin BT (butylthiomethylpenicillin), Penicillin S( ⁇ -chlorocrotylmercaptomethylpenicillin) and Penicillin V(phenoxymethylpenicillin).
• the amount of the substrate which is dissolved in an aqueous suspension of a penicillin amidase-producing microorganism is not critical in the present invention.
• penicillin is dissolved in water at any concentration.
• the aforementioned immobilized microorganism is suspended in the aqueous penicillin solution, and the suspension is stirred. After the reaction is completed, the mixture is filtered or centrifuged to recover the immobilized microorganism for subsequent use. 6-APA is recovered from the filtrate or supernatant solution.
• the optimum condition for complete conversion of penicillin to 6-APA can be readily obtained by adjusting the reaction time.
• the enzymatic reaction of the invention can be performed by a column method. The column method enables the reaction to be carried out in a successive manner.
• the immobilized microorganism is charged into a column, and an aqueous penicillin solution is passed through the column at a suitable flow rate.
• An aqueous solution containing 6-APA is obtained as the effluent.
• 6-APA is recovered by a known method per se such as, for example, adjusting the effluent to a pH of 4 to 5.
• the conversion rate of penicillin to 6-APA mainly depends upon the enzymatic potency of the immobilized microorganism, the temperature or the reaction time. In case of the column method, however, the optimum reaction condition for complete conversion of penicillin to 6-APA can be readily obtained by adjusting the flow rate of the substrate solution.
• the immobilized microorganism of the present invention retains a high level of the enzymatic activity during the reaction. Moreover, due to the sufficient durability of the enzymatic activity thereof, the immobilized microorganism of the present invention can be used repeatedly for the enzymatic reaction.
• the amount of 6-APA in a solution was assayed in accordance with the method described in "Industrial and Engineering Chemistry", 18, 619(1946) and “Analytical Chemistry", 33, 648(1961). That is, a 6-APA solution was adjusted to pH 2.0 with 2N-hydrochloric acid and washed with methyl-isobutylketone to remove penicillin and other contaminants. The amount of 6-APA in the aqueous layer thus obtained was first assayed colorimetrically in accordance with the Iodine Absorption Method. Then, the aqueous layer was further treated with phenylacetyl chloride to convert 6-APA to Penicillin G. The amount of 6-APA in said aqueous solution was again calculated on the basis of the anti-microbial activity thereof which was assayed in accordance with the Paper Disc Method.
• Escherichia coli ATCC 9637 is inoculated into one liter of the medium.
• the medium is cultivated at 30°C for 24 hours under shaking. Then, the medium is centrifuged.
• the microbial cells thus collected show the penicillin amidase activity of 6.5 units/g. 12 g of the microbial cells are suspended in 48 ml of a physiological saline solution. 9 g of acryloylamide, 480 mg of N,N'-methylene-bis-acryloylamide, 6 ml of 5 % ⁇ -(dimethylamino)-propionitrile and 6 ml of 2.5 % potassium persulfate are added to the suspension.
• the suspension is allowed to stand at 37°C for 30 minutes. After the reaction is completed, the suspension is filtered.
• the stiff gel obtained is granulated by passing it through a sieve to form particles of 3 mm in diameter. 120 ml of an immobilized preparation of Escherichia coli ATCC 9637 are obtained. Penicillin amidase activity: 4.7 units/ml.
• An immobilized preparation of Escherichia coli ATCC 9637 is prepared in the same manner as described in Example 1 -- (1). 120 ml of the immobilized preparation are charged into a 2.1 cm ⁇ 34.8 cm column. An aqueous 1 % potassium Penicillin G solution (pH 9.0) is passed through the column at 40°C at a flow rate as shown in Table 1. The percentage conversion of Penicillin G to 6-APA in the effluent is shown in Table 1.
• Escherichia coli ATCC 11105 is inoculated into one liter of an aqueous nutrient medium (pH 7.0) containing the same ingredients as described in Example 1 -- (1).
• the medium is cultivated at 30°C for 24 hours under shaking. Then, the medium is centrifuged.
• the microbial cells thus collected show the penicillin amidase activity of 6.9 units/g. 12 g of the microbial cells are suspended in 48 ml of a physiological saline solution.
• Penicillin V 10 g are suspended in 450 ml of water, and the suspension is adjusted to pH 9.0 with 2N-sodium hydroxide to dissolve Penicillin V therein. Water is added to the resultant aqueous Penicillin V solution to bring its total volume to 500 ml. The, 120 ml of the immobilized preparation of Escherichia coli ATCC 11105 are suspended in the aqueous Penicillin V solution. The suspension thus obtained is stirred for 40°C for a certain period of time. The percentage conversion of Penicillin V to 6-APA in the suspension is shown in Table 2.
• Escherichia coli ATCC 11105 is inoculated into 1 liter of an aqueous nutrient medium (pH 7.0) containing the same ingredients as described in Example 1 -- (1).
• the medium is cultivated at 30°C for 24 hours under shaking. Then, the medium is centrifuged.
• the microbial cells thus collected show the penicillin amidase activity of 6.9 units/g. 12 g of the microbial cells are suspended in 48 ml of a physiological saline solution.
• Streptomyces griseus IFO 3355 is inoculated into one liter of the medium.
• the medium is cultivated at 30°C for 48 hours. Then, the medium is centrifuged.
• the microbial cells thus collected show the penicillin amidase activity of 3.6 units/g.
• 24 g of the microbial cells are suspended in 96 ml of a physiological saline solution.
• 18 g of acryloylamide, 960 mg of N,N'-methylene-bis-acryloylamide, 12 ml of 5 % ⁇ -(dimethylamino)-propionitrile and 12 ml of 2.5 % potassium persulfate are added to the suspension. Then, the suspension is allowed to stand at 37°C for 30 minutes.
• the stiff gel obtained is granulated by passing it through a sieve to form particles of 3 mm in diameter.
• 240 ml of an immobilized preparation of Streptomyces griseus IFO 3355 are obtained.
• Penicillin amidase activity 0.5 units/ml.
• Nocardia gardneri ATCC 9604 is inoculated into one liter of an aqueous nutrient medium (pH 7.0) containing the same ingredients as described in Example 5 -- (1).
• the medium is cultivated at 30°C for 72 hours under shaking. Then, the medium is centrifuged. The microbial cells thus collected show the penicillin amidase activity of 3.6 units/g. 24 g of the microbial cells are treated in the same manner as described in Example 5 -- (1).
• 240 ml of an immobilized preparation of Nocardia gardneri ATCC 9604 are obtained. Penicillin amidase activity: 0.5 units/ml.

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• 1973
  • 1973-03-24 JP JP48033769A patent/JPS5247038B2/ja not_active Expired
• 1974
  • 1974-03-13 US US05/450,655 patent/US3953291A/en not_active Expired - Lifetime
  • 1974-03-20 SE SE7403767A patent/SE431768B/xx not_active IP Right Cessation
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  • 1974-03-22 CH CH407074A patent/CH593980A5/xx not_active IP Right Cessation
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